Sequence analysis, prokaryotic expression and antiserum preparation of coat protein gene of Actinidia virus A in Sichuan province

Abstract: 【Objective】The purpose of the research was to study the occurrence and molecular diversity of Actinidia virus A(AcVA), so as to provide scientific basis for the rapid detection, scientific prevention and control of AcVA and the variety breeding of kiwifruit in China.【Methods】Total RNA was extracted from kiwifruit leaves by CTAB(Cetyl Trimethyl Ammonium Bromide). Nested-PCR was used to amplify AcVA CP gene sequence. The first round PCR amplification was performed by using primers AcVA-1 F(5'-ATGAATCGTTCGAGCA TAGGT-3') and AcVA-1 R(5'-TGCGAACATGGTCCCACACTTA-3'), and the pair of primers was designed according to the full length of AcVA sequence, and the amplified fragment was 888 bp that included the complete CP gene sequence. The reaction was conducted under conditions of initial 5 min denaturation at 94 ℃, 34 cycles of 94 ℃ for 60 s,54 ℃ for 60 s, 72 ℃ for 60 s and extension for 10 min at 72 ℃. Then, 1 μL PCR product was used as template for the second round of PCR amplification with the primers AcVA-2 F(5'-ATGGCAAAGAATATCTCAAG-3') and AcVA-2 R(5'-CTATATTTCAACAGCCTGC-3'). PCR was performed using the following parameters: one cycle at 94 ℃ for 5 min, 34 cycles at 94 ℃ for 30 s, 54 ℃for 30 s, and 72 ℃ for 40 s, and extension for 10 min at 72 ℃. The BLAST algorithm was used to search the NCBI GenBank(http://www.ncbi.nlm.nih.gov/) databases for homologous sequences and ascertain the identity of target gene. DNAMAN was used to analyze the AcVA CP gene sequence, and MegAlign was used to analyze the sequence identity. The phylogenetic tree was constructed using the Neighbor-Joining(NJ) method in MEGA 6.0. The restriction enzymes Bam HⅠ and Sal Ⅰ were added to the corresponding end of the AcVA CP gene sequence, respectively. The PCR purified products and pET28 a vector were digested by Bam HⅠ and Sal Ⅰ, after that they were ligated with T4 DNA ligase(TaKaRa) and transferred into E. coli(Escherichia coli) strains DH5á(TaKaRa), and finally plated onto LB(Luria-Bertani) agar containing Kana(Kanamycin). The expression strain BL21 containing the recombinant plasmid was cultured at 37 ℃ overnight, and transferred to a new medium at a 10% inoculum on the second day, until OD600 reached 0.4-0.6, IPTG(Isopropyl β-D-Thiogalactoside) was added to a final concentration of 0.2, 0.4, 0.6, 0.8, 1.0 mM, and incubated at 16 ℃ overnight. The expressed protein was purified and then anti-AcVA-CP antibody was obtained from a rabbit. The optimal titer of the antiserum was tested by Western blot.【Results】The complete CP gene sequences of AcVA Sichuan isolates were cloned by nested-PCR and named as AcVA-DJY4, AcVA-HYZ1 and AcVA-WBS12, respectively. In the study the frequency of AcVA in Sichuan province was also counted. The full length of the CP gene sequence was 597 bp, encoding 198 amino acids. Based on the comparison of the nucleotide sequence and the deduced amino acid sequence of CP gene of the AcVA isolates, the nucleotide identity between the AcVA isolates from Sichuan province ranged from 86.9% to 88.3%, and the identity of the encoded amino acids ranged from 96.0 to 96.5%. The CP genes of three AcVA Sichuan isolates shared87.9%-90.8% nucleotide identity and 94.9%-97.5% amino acid identity with the Actinidia virus A isolate TP7-93 A(AcVA-TP7-93 A) from New Zealand and the Actinidia virus A isolate Haenam(AcVAHaenam) from South Korea. Phylogenetic analysis based on nucleotide sequence of AcVA CP gene showed that AcVA-HYZ1 was clustered with AcVA-Haenam(Group I), and AcVA-WBS12 was clustered with AcVA-TP7-93 A(Group II), but AcVA-DJY4 was an independent branch. Phylogenetic analysis based on amino acid sequence of AcVA CP gene showed that AcVA-WBS12 was a single branch,AcVA-DJY4 and AcVA-HYZ1 were clustered into one group(Group I), and the AcVA-Haenam reported in South Korea and the AcVA-TP7-93 A reported in New Zealand were clustered into another group(Group II). The results of the phylogenetic analyses indicated that there were significant differences among the AcVA isolates. The prokaryotic expression plasmid pET28 a-AcVA-DJY4-CP was successfully constructed. The target fusion protein(27 kDa) was highly expressed in E. coli induced by 0.6 mmol·L^-1 IPTG at 16 ℃. The expressed protein was purified and retrieved and then used to immune rabbits and the corresponding specific antiserum was prepared. Western blot analysis confirmed that the antiserum reacted strongly and specifically with the CP of AcVA-DJY4, with the optimal titer of the antiserum being up to 1:5 000.【Conclusion】Three AcVA Sichuan isolates were obtained for the first time, enriching the sequence diversity of the AcVA CP gene sequences. In the study, the optimal conditions were explored for prokaryotic expression and specific antisera was prepared for detection of AcVA-DJY4-CP. Western blot analysis showed that the antiserum could be used for the detection of AcVA in the kiwifruit producing districts. These results can also provide a technical support for the rapid detection, scientific prevention and control of virus disease in kiwifruit plant and the breeding of kiwifruit varieties.